1. Field of the Invention
The present invention relates to compounds and methods for modulating transcription activated by β-catenin/TCF, for example, the selective inhibition of genes targeted by the Wnt/β-catenin pathway.
2. Description of the Related Art
The Wnt/β-catenin pathway initiates a signaling cascade critical in both normal development and the initiation and progression of cancer (Wodarz et al., “Mechanisms of Wnt signaling in development,” Annu. Rev. Cell Dev. Biol. 14:59-88 (1998); Morin, P. J., “Beta-catenin signaling and cancer,” Bioessays 21:1021-30 (1999); Moon et al., “The promise and perils of Wnt signaling through beta-catenin,” Science 296:1644-46 (2002); Oving et al., “Molecular causes of colon cancer,” Eur. J. Clin. Invest. 32:448-57 (2002)). The hallmark of this pathway is that it activates the transcriptional role of the multifunctional protein β-catenin. In normal cells, the majority of β-catenin is found at the cell membrane bound to cadherin where it plays an important role in cell adhesion. Another pool of β-catenin is found in the cytoplasm and nucleus where it regulates transcription (Gottardi et al., “Adhesion signaling: how beta-catenin interacts with its partners,” Curr. Biol. 11:R792-4 (2001)). In its diverse roles as a mediator of cell adhesion at the plasma membrane, and as a transcriptional activator, β-catenin interacts with a host of proteins, the majority of which, despite a lack of significant sequence homology, compete for the same armadillo-repeats of β-catenin. The crystal structure along with mutational studies has mapped the β-catenin binding sites of several proteins to various armadillo repeats (Gottardi et al., “Adhesion signaling: how beta-catenin interacts with its partners,” Curr. Biol. 11:R792-4 (2001); Huber et al., “The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin,” Cell 105:391-402 (2001)).
The cytoplasmic pool of β-catenin is regulated via phosphorylation by the “destruction complex” that includes glycogen synthase kinase-3β (GSK-3β), casein kinase-1α (CK-1α), the scaffold protein, Axin, and the tumor suppressor, adenomatous polyposis coli (APC), among others (Behrens J., “Control of beta-catenin signaling in tumor development,” Ann. N.Y. Acad. Sci. 910:21-33 (2000); discussion 33-5). In the absence of Wnt signaling, phosphorylation marks the cytoplasmic β-catenin for Skp1-Cullin-F box (SCF)-directed ubiquitination and proteosomal degradation. Activation of the Wnt pathway inactivates the function of GSK-3β, preventing β-catenin phosphorylation, thereby allowing β-catenin to accumulate in the cytoplasm and subsequently translocate to the nucleus where it forms a transcriptionally active complex and drives the expression of its target genes. A key step in the activation of target genes is the formation of a complex between β-catenin and members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF-1) family of transcription factors (Brantjes et al., “TCF: Lady Justice casting the final verdict on the outcome of Wnt signaling,” Biol. Chem. 383:255-61 (2002)). To generate a transcriptionally active complex, β-catenin recruits the transcriptional coactivators, CREB-binding protein (CBP) or its closely related homolog p300 (Hecht et al., “The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates,” EMBO J. 19:1839-50 (2000); Takemaru et al., “The transcriptional coactivator CBP interacts with beta-catenin to activate gene expression,” J. Cell Biol. 149:249-54 (2000)) as well as other components of the basal transcription machinery.
The precise mechanism by which the β-catenin/TCF complex activates transcription of Wnt responsive genes is not clear, but domains of β-catenin involved in transcriptional activation have been mapped to the NH2- and COOH-termini (Staal et al., “Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin,” EMBO 3:63-68 (2002)). The COOH-terminal region of β-catenin consists of approximately 100 amino acids and it has been shown to interact with the TATA binding protein (TBP) (Hecht et al., “Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA-binding protein in vitro,” J. Biol. Chem. 274:18017-25 (1999)). When fused to LEF-1, the COOH-terminus is sufficient to promote transactivation (Vleminckx et al., “The C-terminal transactivation domain of beta-catenin is necessary and sufficient for signaling by the LEF-1/beta-catenin complex in Xenopus laevis,” Mech. Dev. 81:65-74 (1999)). The NH2-terminal portion of β-catenin consists of approximately 130 amino acids containing the GSK-3β phosphorylation sites required for proteosomal degradation.
The Wnt/β-catenin pathway normally regulates expression of a range of genes involved in promoting proliferation and differentiation. However, in >85% of colon cancers one of the components of the destruction complex, APC, and/or β-catenin itself is mutated, leading to an increase in nuclear β-catenin and constitutive activation of target genes (Fearnhead et al., “Genetics of colorectal cancer: hereditary aspects and overview of colorectal tumorigenesis,” Br. Med. Bull. 64:27-43 (2002)). Many of these genes, including cyclin D1 (Shtutman et al., “The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway,” Proc. Natl. Acad. Sci. USA 96:5522-27 (1999); Tetsu et al., “Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells,” Nature 398:422-26 (1999)) and c-myc (He et al., “Identification of c-MYC as a target of the APC pathway,” Science 281:1509-12 (1998)) which play critical roles in cell growth, proliferation, and differentiation, together with genes necessary for invasive growth like matrilysin (Crawford et al., “The metalloproteinase matrilysin is a target of beta-catenin transactivation in intestinal tumors,” Oncogene 18:2883-91 (1999)), fibronectin (Gradl et al., “The Wnt/Wg signal transducer beta-catenin controls fibronectin expression,” Mol. Cell. Biol. 19:5576-87 (1999)), CD44 (Wielenga et al., “Expression of CD44 in Apc and Tcf mutant mice implies regulation by the WNT pathway,” Am. J. Pathol. 154:515-23 (1999)), and μPAR (Mann et al., “Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas,” Proc. Natl. Acad. Sci. USA 96:1603-08 (1999)) are inappropriately activated.
Given that the majority of colorectal cancers involve activation of the β-catenin signaling pathway, and given the fact that multiple mutations lead to this activation, there is a clear need for drugs that attenuate the nuclear functions of β-catenin. The present invention provides agents which antagonize β-catenin/TCF-mediated transcription, and provides further related advantages as described in detail below.